Second generation of Cord Blood Banks

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1 Second generation of Cord Blood Banks Dr Sergio Querol, Dra Marta Torrabadella, Dr Joan Garcia, Prof Alejandro Madrigal Anthony Nolan Research Institute, London & Programa Sang de Cordó, Barcelona

2 QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (LZW) decompressor are needed to see this picture. Blood, 1974, Vol. 43, No. 3, pp QuickTime and a TIFF (LZW) decompressor are needed to see this picture. QuickTime and a TIFF (LZW) decompressor are needed to see this picture.

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4 BM versus UCB in Adults Rocha et al. NEJM 2004; 351:2276 Laughlin et al. NEJM 2004; 351:2265 Not so good for UCB why? 1) low cell dose 2) 2 antigen HLA mismatched 3) learning curve

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8 Cord blood banks: The research phase Clinical success of first generation of cord blood banks: The Eurocord/Netcord synergy Clinical assessment and advice Quality (International standards)

9 But, what is a Cord Blood Bank? QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. 2001: Standards for Cord Blood Services

10 QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

11 Threats Efficiency: <30% collected, stored <1% stored, transplanted Financial: New limits, no break-even Continuously increasing transplant costs

12 Collection/transport:~ units collected, 470,000 1 billion cells? Discard Processing Fix cost:~ % 90,000 Clinical Accredited process Processing/Testing: ~ % 380,000 First generation of cord blood banks Break-even (1%)= 47,000 Public funding= National programmes Charitable funding

13 Questions Which size? Cost-benefit study How do we get this size promptly? Financial engineering How do we obtain the most of cord blood? Pharmaceutical approach

14 Opportunities Excellent biological profile Non-questioned ethical advantage Social awareness Individual demand Stem cell revolution: grants Research needs: partnerships Non-personalised cell therapies: new blood bank model

15 Public Collection Programmes Private Services Cord Blood Factory Allogeneic Bone Marrow Register BMT units: CBT DLI CordPharm Academia and Industry: Research Bio-resource Non-personalised Cell Therapies

16 Public Collection Programmes Private Services Cord Blood Factory Allogeneic Bone Marrow Register BMT units: CBT DLI CordPharm Academia and Industry: Research Bio-resource Non-personalised Cell Therapies

17 The role of perinatal care providers J Perinat Med. 2007, 35(4): Umbilical cord blood collection: do patients really understand? Fox NS, Stevens C, Ciubotariu R, Rubinstein P, McCullough LB, Chervenak FA. Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, USA. Women are poorly informed about cord blood banking. The decision making process should be conducted with the goal of ensuring every pregnant woman the opportunity to make a well informed decision about cord blood banking The women s dilemma To transfer or to maintain ownership? QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. But Universal access to private donation but 2000 Reduced access to public donation (<1% population)

18 New requirements for Women Services Integration of cord blood donation as a service in a Women s Service: new role for Obstetricians and Midwives. Model: Public vs private Workload Safety: In utero vs Ex utero Requirements in terms of Equipment, HRs and Training License for procuring: Private banking: service fee Public banking: Institutional funding/third party agreement

19 Cord blood donation: activities Education Training Informed Consent Collection Non cryopreserved Storage Non cryopreserved Transport Follow up

20 Close Single Capital, 0.15M: HRs: 2 IT Collection trolley Blood bank Transport High collection profile Low repertoire, low thresholds Low growth

21 Close Multiple: CONCORDIA model Intermediate collection profile Good repertoire, high thresholds Intermediate growth Funding?

22 public maybe ~ 200 (coll+transp) Open: new public/private partnerships Similar that adult bone marrow registers General campaign Access to any mother interested in public donation Require explicit collaboration Perinatal Care Provider Potentially funded by donor (Virgin Health Bank model or Stemcyte model) as a service or as a gift Low collection profile Good repertoire, low thresholds High growth Funding: private by fees (~ 2,000)

23 Public Collection Programmes Private Services Cord Blood Factory Allogeneic Bone Marrow Register BMT units: CBT DLI CordPharm Academia and Industry: Research Bio-resource Non-personalised Cell Therapies

24 The Anthony Nolan Cell Therapy Centre Factory (reception, processing, storing, dispatch) Lab (safety, identity, potency) Quality (Quality Control and Quality Assurance)

25 Towards a Cell Factory Concept: Critical issues 1. Fresh storage and transport 2. Automation 3. Testing: Safety/Identity/Potency 4. Quality assurance: The Anthony Nolan Cord Blood Database

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27 FACILITY -200 m 2 plant-building, three floors: factory, laboratory, technical plant -106 m 2 for Production, 73 m 2 for quality control and 83 m 2 for administration Ground floor: C Sepax/CoolMix Manufacture CBIS CRF Cryoservice/ MVE/Chart D 3k 3k Capacity: 24,000 units 1 research tank 1 permanent quarantine 3 clinical tanks CliniMacs 6k Productivity: Reception FMS Distribution Reference samples Fluidx Cryogenics 6k 6k 2000/ per shift 1 Manager 3 Supervisors 4 Technicians Fix costs: 0.5M/year

28 Reception Distribution Manufacturing Cryopreservation Long-term storing

29 Anthony Nolan Cord Blood Database

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31 Releasing tests Potential risks: Safety Identity Potency What to do: Built quality in front Verify: releasing tests Safety: -Serology on cord blood Identity: -HLA -Blood group and gender - Maternal haplotype Potency: -CFU/CD34/CLONE -Viability -NC recovery -Volume

32 PREDICTIVE UTILITY OF THE ATTACHED SEGMENT IN THE QUALITY CONTROL OF A CORD BLOOD GRAFT. A ) CD 34 / 10 3 TNC n=20 CD34 ATTACHED SEGMENT 3,0 2,5 2,0 1,5 1,0 0,5 0,0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 UCB BAG Y = 0,930 x + 0,008. R 2 =0,85 CFU B ) ATTACHED SEGMENT CFC / 10 5 TNC n= UCB BAG Y = 0,540 x + 29,130. R 2 =0,78 CLONE C ) ATTACHED SEGMENT ECLONE n= UCB BAG Rodriguez L, Garcia J and Querol S. Biol Biol Blood Marrow Transpl 2005 Y = 0,711x + 10,31. R 2 =0,83

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34 CD34+ CELLS FROM CORD BLOOD UNDERGO APOPTOSIS IN CERTAIN CONDITIONS AAD SSC-H: Side Scatter Annexin V AAD

35 Public Collection Programmes Private Services Cord Blood Factory Allogeneic Bone Marrow Register BMT units: CBT DLI CordPharm Academia and Industry: Research Bio-resource Non-personalised Cell Therapies

36 GERMAN ADULT INVENTORY: ~3,000,000 % 2 months (urgent): 50% % 4 months (non-urgent): 75% Searches duration for German patients Source: Annual report , ZKRD Cord blood programmes are no critical 1 unrelated TX / inhabitants ~5% from cord blood

37 40% from cord blood Spain (~60,000 adult donors): probability to find a donor 7 or 8 out of 8 % 2 months (urgent): 25% % 4 months (non-urgent): 45% Cord blood programmes are critical 0.5 unrelated /100000

38 TOWARDS AN EQUITABLE ACCESS TO A TIMELY THERAPY sibling 1/ The complementary approach 4/6 5/6 6/6 HLA restriction 1/ Acceptable INCLUSIVE Optimal RESTRICTED unrelated Target: 3/ PLACENTAL TYPE thousands National sufficiency ADULT TYPE millions International collaboration

39 Graft Identification Advisory Service (GIAS) Allo-Mandatory Search Score (HLA)? Optimal date for transplant? shift Best Adults Best Cords Objective: 100% transplants on time

40 Impact of cell dose and HLA Match on survival. Data presented by Dr Pablo Rubinstein at the 5 th International Umbilical Cord Blood Symposium held in Los Angeles in May Survival 5/6 Match 4/6 Match 5/6 match 4/6 match Cell Dose ( 10 7 /kg) 70% >10 50% % < <2.5 time after transplant Barker J et al. The Dose-Match Interaction in Umbilical Cord Blood (UCB) Transplantation: An Analysis of the Impact of Cell Dose and HLA-Match on the Disease- Free Survival (DFS) of 989 Patients Transplanted with Single Units for Hematologic Malignancy. Blood (ASH Annual Meeting Abstracts) : Abstract 333

41 How big should the cord blood bank be? Probability to find a least 1 HLA-A,B low and DRB1 high match All patients 2,000 One road, three stations: Non north western European patients 722 Querol et al, Haematologica in press -Institutional bank: 4/6; 10,000; 90% -National programme: 5/6, 50,000; 80% -International network: 6/6; 1,000,000; 70%

42 Public Collection Programmes Private Services Cord Blood Factory Allogeneic Bone Marrow Register BMT units: CBT DLI CordPharm Academia and Industry: Research Bio-resource Non-personalised Cell Therapies

43 Cord blood bank model: the classical blood bank Development of minimally manipulated cord blood components Liaison with Biotech companies for advanced cell therapies CordPharm by-products CELLS: 1. CBMC emnc (Biosafe) vs Manual Ficoll (Laura) 2. PROGENITOR CELL POOLS CD133 selection (Roger, Richard) 3. T REG POOLS CD25 selection (Daniel, Richard) 4. T EFFECTOR ALLODEPLETION CD137 vs CD25 depletion (Jim) 5. NK ALLOREACTIVE CELLS CD3 depletion and NK expansion (Mehri) HAEMODERIVATIVES: 1. PPP MALDI (Sergio) 2. PRP MSC isolation and expansion (pendent) 3. RBC (Foetal Hgb) Marta

44 RESEARCH IN A CORD BLOOD BANK The basis of our research is finding what is unique in cord blood and then test their abilities for therapy Therefore, our research model is to assess the differences between the blood components of newborns vs adults by: Pheno(geno)tipyc characterisation Functional assays Then, the bank acts as catalyst for translational research linking with scientists working in cell therapy and providing clinical grade products for their pre-clinical work

45 The Anthony Nolan Research Institute Cellular Immunotherapy Group Prof Alejandro Madrigal, Dr Sergio Querol

46 Predicting function using a flow cytometry modified ISHAGE strategy to enumerate CD34: annexin V-7AAD costaining Richard Duggleby PhD

47 ISHAGE gating of stem cells favors the enumeration of live cells COM15 freshly isolated CD34+ cells; final lymphocytes ISHAGE gate (CD34+CD45low) COM15 thawed CD34+ cells; final lymphocytes ISHAGE gate (CD34+CD45low) SSC-H: Side Scatter SSC-H: Side Scatter FSC-H: Forward Scatter FSC-H: Forward Scatter Thawing the same sample shows that the ISHAGE gate favors live cells. But whilst dead cells might be removed there still maybe apoptic cells

48 Use of Annexin V to further characterize the ISHAGE gated population T=2 days 7AAD Following Fresh cord blood samples with time shows how the stem cell population can become apoptic whilst still having a low 7AAD count Annexin V T=7 days 7AAD Annexin V

49 Frozen cords can have a high degree of variability Transplanted cord successful engraftment 7AAD Assessment with Annexin V and 7AAD of the ISHAGE gated stem cells reveals that there can be a significant number of apoptic cells present post thaw of cord units. An Annexin V mid viability frozen cord 7AAD An Annexin Low viability frozen cord 7AAD Annexin

50 Viable stem cell assessment predicts the CFU of a sample Live counts Ishage CFU/ul 50 Pearson r R squared P value (two-tailed) Pearson r R squared P value (two-tailed) CFU/ul live CD34+/ul ishage 34/ul Assessment of stem cell numbers in fresh cord blood, frozen CBMC and and frozen cord blood samples; Stem cell numbers determined using ISHAGE gating (with 7AAD) and adjusted using assessment utilizing Annexin V staining as well. Recent evidence correlates CFU of cord samples with engraftment. Here, by adjusting the ISHAGE gated stem cell count using the viable stem cell count assessed we can better predict the CFU.

51 High purity and yield of natural Tregs from cord blood usning a single step selection method Dr Daniel Figueroa-Tentori

52 New method

53 Gated on CD4+ cells CD CD CD CD127 MidiMacs isolation CD4

54 Phenotype post-isolation 90% correlation with FOXP3+ gating on CD25 high CD127 low Seddiki, N. J.Exp. Med.2006; 203:1701

55 Tregs repertoire Pool Ag specific Polyclonal expansion Less cells needed Auto or donor specific Known Ag Skewed repertoire FOXP3 demethylation Auto or donor specific Time consuming Unknown Ag Maintain starting repertoire Adult = Rapamycin Practical starting cell count Third party Unknown Ag Expand if needed No need of Rapamycin

56 CliniMACs isolation of CD25+ CB pools: product characteristics and functional assessment Richard Duggleby PhD

57 Purity of CliniMACS isolations CD eff CD CD CD Treg CD25 Purity COM CD25 Treg/Eff ratio com com10 com11 com12 COM5 7 6 % of CD45+ cells Treg/eff %CD25+ Treg %CD25+Eff %cd25- Treg %CD25- eff 0 com10 com11 com12 COM5 Initial CD25+ isolations (COM5) were heavily contaminated with CD127hiCD25+ effector cells; giving a low Treg to effector T cell ration in the final product. By using lessons learnt using the MidiMACS columns we were able to translate method changes (in press) to the CliniMACS. Despite the increased purity still 6-11x10e6 cells

58 Cord CD25+ cells isolated using CliniMACS Ab can suppress a polyclonal stimulus when in combination Cord CD25+ cells isolated on the MidiMACS have comparable purity. 700 Response Individual Cords to anti-cd3/cd28/cd2 inspector beads 120 % response by combine cords An206 (CORD B) An204 (CORD A) 100 %response suppression 40 AN204 (A) AN206 (B) : :0 1:0 1:1 0:1 No. of cells/well (x10e6) Ratio eff:treg combined A+B no. of CD25+ added to CBMC-25 (A+B) Initial experiments (needs repeating). One of the cords responded poorly to inspector beads but in combination both responded and were suppressed by individual or combined CD25+ cells. Need to repeat using the CliniMACS

59 Treg function +1 day post isolation Suppression vs Cord CD2 Suppression vs Cord CD %response COM11 COM10 COM12 cpm COM11 COM10 COM :0 1:1 0 1:0 1:1 After O/N transportation cord CBMC-25 responded poorly to inspector beads and thus only a trend of suppression is seen by cord CD25+ cells. Also poor suppression of adult PBMCs responses to beads was also observed; cord CD25+ cells have in the past suppressed bead adult PBMC responses.

60 Allodepletion of Cord T cells with CD25 / CD137

61 CD25- CBMCs as a source for DLI The Anthony Nolan Cord Blood bank is working on providing CD25 + Tregs as a therapeutic resource for the suppression Of GvHD. The CD25- CMBCs could be used as a source for DLI with the prior selected CD25 T regs added back to the DLI after allo depletion. Working with CD25- CBMCs as a starting resource could enable the use of CD25 as a target for allo depletion.

62 CD25 APC CD25 / CD137 of CD25 depleted CBMCs in MLR with PBMCs CD137 PE FL2-H: CD137 PE FL2-H: CD137 PE FL4-H: CD25 APC FL2-H: CD137 PE FL2-H: CD137 PE FL4-H: CD25 APC FL2-H: CD137 PE FL2-H: CD137 PE FL4-H: CD25 APC FL4-H: CD25 APC FL4-H: CD25 APC FL4-H: CD25 APC

63 CD25/CD137 expression in proliferating cord T cells CD3/CD28 Dynal bead stimulation of CFSE labeled CD25 depleted CBMCs CD3 PerCP FL3-H: CD3 PerCP CD25 APC FL4-H: CD25 APC CD137 APC FL4-H: CD137 APC FSC-H FL1-H: CFSE FL1-H: CFSE FSC CFSE

64 Cord blood NK cells Mehri Daryouzeh

65 CD56 CD16 expression in isolated CBMCs NK cells QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

66 Cord Blood CD cells are possible precursors of mature natural killer cells or a new lineage of NK cells? CD16-CD56+ CD117 bright CD94 + NKp30 + 2B44 + CD CD16+CD56+ CD117 dim CD94 + NKp30 + 2B CD16 CD16+CD56- CD117- CD94 dim NKp30 dim 2B44 +

67 ~ units collected, 530,000 ~10% 1 billion cells? 90, million cells? Reagent ~ % Clinical Accredited process Research Basic clinical grade ~ 400 ~ % 40% 380,000 60,000 Increasing efficiency: -all units collected, used -research supply -new applications --low extra investment 2nd generation of CBB

68 ANTHONY NOLAN CORD BLOOD PROGRAMME QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Susana Garcia Gomez Robert Davy Roger Horton Laura Fry Daniel Gibson Salmah Mahmood Hazel Forde Richard Duggleby James Devitt Daniel Figueroa-Tentori Lia Zambetti Mehri Daryouzeh Dr Nichola Cooper (ICH) Prof John Martin (UCL) Prof Manuel N Fernandez (CPH, Madrid) Prof Robert Rees QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Prof Tony Dodi Dr Sergio Querol Prof John Goldman Prof Alejandro Madrigal Prof Ghulam Mufti Dr Joan Garcia Dra Marta Torrabadella QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

69 ONE BIRTH, MULTIPLE LIFES Thank you for your attention

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